PROJECT ABSTRACT Foxp3+ regulatory T (Treg) cells are critical for the regulation of immune responses to self, foreign, and tumor- associated antigens, prompting considerable interest in the therapeutic manipulation of Treg cells in the context of cancer, autoimmunity, and transplantation. Over the past decade, a paradigm has emerged holding that thymic-derived Treg (tTreg) cells recognize self antigens. Furthermore, substantial evidence indicates that antigen recognition is critical for many aspects of Treg biology, including development, activation, anatomical distribution, and suppressor function. However, the natural self antigens recognized by tTreg cells remained unidentified. Without this knowledge, it was not possible to determine the biochemical nature of antigen recognition by Treg cells, to elucidate the role of TCR signal strength in promoting Treg development and clonal deletion, and to directly characterize endogenous antigen-specific Treg populations using peptide/MHC multimers. In recent work, we have identified a self antigen recognized by an endogenous population of naturally occurring Treg cells. The antigen, referred to as ?C4? in this proposal, is an unmodified I-Ab-restricted peptide derived from a prostate-specific protein, and is recognized by a canonical Treg clone named ?MJ23?. The studies outlined in this proposal will utilize the MJ23 TCR:C4/I-Ab system to address long-standing critical gaps in knowledge regarding Treg biology and the mechanisms by which dominant and recessive tolerance are enforced. The objectives of this proposal are to elucidate the biochemical nature of pMHC recognition by tTreg TCRs, to identify the mechanisms driving the thymic development of C4-specific Treg cells, and to elucidate the processes coordinating the peripheral homeostasis of C4-specific Tregs. It is our central hypothesis that high-affinity recognition of C4/I-Ab drives the thymic development of C4-specific Treg cells and the further selection of optimal C4-specific Treg clones in the periphery. We will test our central hypothesis and accomplish the objectives of this application by pursuing the following specific aims. In Aim 1, we will elucidate the molecular basis of Treg TCR recognition of self pMHC. In Aim 2, we will identify the signals driving the thymic development of C4-specific Treg cells. In Aim 3, we will determine the impact of peripheral selection on C4-specific Treg cells. The work outlined in this proposal is expected to reveal the biochemical nature of self antigen recognition by naturally occurring tTreg specificities. Moreover, the studies are expected to elucidate whether TCR signal strength is a primary determinant of whether self-reactive thymocytes will differentiate into the Treg lineage or undergo clonal deletion. Finally, these studies are expected to reveal the extent to which antigen encounter in the periphery shapes the repertoire of antigen-specific tTreg cells throughout life. In all, our work is expected to yield new insights into Treg cell biology and the mechanisms by which host tissues are protected from autoimmune attack.